Thiamin is an essential component of the human diet and is added to many commercial foods (RDA = 1.4 mg). Deficiency in this vitamin results in a neurological disease called beri-beri. At the molecular level, thiamin dependent enzymes play a particularly important role in carbohydrate metabolism and include transketolases, alpha-ketoacid decarboxylases, alpha-ketoacid oxidases and acetolactate synthase. The long range goals of our research is the complete understanding of the mechanistic enzymology of thiamin biosynthesis in Escherichia coli from a mechanistic and structural perspective. These studies are significant for three reasons. Firstly, since this vitamin is a required component of the human diet, it is important to understand how it is biosynthesized. Secondly, from the perspective of basic science, the biosynthetic pathway involves an unusually large amount of novel mechanistic chemistry. Lastly, our studies will facilitate the construction of overexpression strains that may be of use for the commercial production of thiamin or its components (2,000 tons/year produced by total synthesis). In this grant period, we will complete our mechanistic studies on thiamin phosphate synthase and on thiaminase I. In parallel, we will carry out a series of experiments designed to identify the reactions involved the sulfur transfer from cysteine to the acyl adenylate of a 66 amino acid carrier peptide. The reconstitution of the thiazole assembly from tyrosine, 1-deoxy-D-xylulose-5-phosphate will also be investigated using overexpressed ThiFSGH and ThiI. Once we have identified specific reactions in the sulfur transfer and in the thiazole formation, we will initiate systematic mechanistic studies on each of the enzymes involved.